Endothelial cells form intercellular connections (tight junctions) to limit permeability of the vessels that these cells line. However, these junctions are regulated to allow permeability to increase in response to specific signals or to allow vessel growth or repair. The activity of the guanosine triphosphatase RhoA is critical to the stability of tight junctions; too little or too much RhoA activity increases paracellular permeability. Ngok et al. identified Syx in a screen of Rho guanine nucleotide exchange factors (Rho GEFs) as a protein that colocalized with tight junctions in human umbilical vein endothelial cells (HUVECs) and the epithelial cell line MDCK. Endogenous and exogenously expressed Syx coimmunoprecipitated and colocalized with Mupp1, a tight junction scaffold protein, and with several other components of the CRB (Crumbs-Pal-Patj) polarity complex. Knockdown of Syx disrupted the actin cytoskeletal circumferential ring in HUVECs, reduced the localization of VE-cadherin at sites of intercellular contact in HUVECs and neonatal human dermal microvascular endothelial cells (HMVECs), and reduced transendothelial impedance (a measure of the “tightness” of intercellular tight junctions) of HUVECs. Mice deficient in syx (syx–/–) have an angiogenesis defect, and endothelial cells from these mice exhibited reduced transendothelial impedance in culture, and the mice had increased vascular permeability in a microsphere extravasation assay and a skin dye assay and increased cardiac stiffness likely due to cardiac edema. Using a combination of pharmacological inhibitors, constitutively active constructs, and knockdown experiments, the authors identified Diaphanous, which inhibits VE-cadherin endocytosis and Src activity; Src; and ROCK as proteins downstream of Syx. Angiopoietin-1 (Ang1) and vascular endothelial growth factor A (VEGF-A) strengthen and disrupt tight junctions to promote or reduce endothelial barrier function, respectively. Ang1 promoted the recruitment of Syx to tight junctions, whereas VEGF-A reduced Syx at cell junctions at a time that coincided with irregular patterns of other tight junctional proteins. The ability of Ang1 to increase transendothelial impedance was lost if Syx was knocked down, and its ability to inhibit dye leakage from the blood vessel into the skin was lost in syx–/– mice. VEGF-A also failed to increase dye leakage in syx–/– mice. Thus, VEGF-A and Ang1 signals appear to converge on Syx. The phorbol ester PMA and VEGF-A, both of which activate protein kinase D (PKD), cause similar effects on tight junctional proteins and Syx localization. Syx phosphorylation at a PKD motif was increased by VEGF or PMA, and expression of Syx with a mutation of this site prevented the VEGF-A–mediated alterations in Syx localization at cell junctions. The authors propose that vessel permeability signals converge at Syx, the abundance of which at the junctional complex is reduced by phosphorylation, to regulate the activity of RhoA at tight junctions and thereby control endothelial cell permeability.